The long-term goal of this project is to understand the molecular mechanism of human mismatch repair (MMR) and its impact on human health and disease. The importance of MMR is underscored by the fact that defects in MMR genes lead to genomic instability and eventually to cancer predisposition. Despite the fact that the excision-step of the human MMR reaction has been recently reconstituted using purified proteins, including MutS alpha, MutL alpha, EXO1, and RPA, many fundamental questions in MMR still remain unanswered. For example, how mismatch-provoked excision initiates and terminates, and what role MutL alpha plays in MMR are unknown. Through the reconstitution of the 5' nick-directed human MMR reaction, we found that MutL alpha negatively regulates mismatch-provoked, EXO1-catalyzed excision and terminates it immediately upon mismatch removal. Given the dependency of the EXO1-catalyzed mismatch excision on MutS alpha and physical interactions among EXO1, MutS alpha, and MutL alpha, we hypothesize that mismatch-provoked excision is precisely regulated through interactions among these proteins. Specifically, in the presence of a mismatch, MutS alpha preferentially interacts with EXO1 to promote excision; but the removal of the mismatch by EXO1 allows EXO1 to preferentially interact with MutL alpha, which then terminates the excision. To test this hypothesis, a series of EXO1, MutS alpha, MutL alpha mutant proteins that disrupt the interaction of EXO1 with one Mut protein but not the other will be constructed, expressed, and purified. These mutant proteins will be tested in an in vitro reconstitution system for their ability to perform mismatch-provoked excision at the initiation and at the termination. Analysis of the excision intermediates in each reaction will reveal how the interactions among MutS alpha, and MutL alpha, and EXO1 regulate a particular step of the excision reaction. Finally, to understand how efficiently the MMR system terminates mismatch-provoked excision, the excision termination sites for heteroduplexes with different sequence contents and distances separating the mismatch and the strand break will be precisely determined. In addition to revealing the molecular mechanism of mismatch-provoked excision, this study will provide important clues to understand many puzzling phenomena in mlh1 null mutants regarding 5' directed MMR, meiotic cell apoptosis, and anti-recombination.